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Figure 3 supplement 1. Presence of spinal Islet1 and ChAT mRNAs at different
developmental stages. In situ hybridization of the two mRNAs was performed with the same
protocol (see Methods). The Islet1 probe was synthesized according to Shi et al. (2009) and
involved amplifying a PCR fragment of 798 pb by the primers 5’-
AAGTGCAACATCGGCTTCAG-3’ and 5’-GCTGTTTGGGGTATCTGGGA-3’. Despite the
occasional appearance of a widespread background signal (due to differences in colorimetric
reaction times), Islet1 mRNAs in darkened clusters were clearly evident in both the axial and
appendicular motor columns at all developmental stages examined (stage 51 (upper left panel)
and 55 (lower left panel) are illustrated), whereas ChAT mRNAs were found in limb MNs
only from stage 55 onwards (c.f., upper and lower right panels). |
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Figure 3. Motoneuronal identification of non-cholinergic limb projecting neurons.
A. Example of fluorescence immunolabeling against ChAT (magenta) and Islet1/2 (orange) in
appendicular and axial MNs (Ap. MN, Ax. MN) in a stage 52 tadpole. Appendicular MNs
were previously labeled with retrograde Alexa Fluor dextran 647 (AD 647, cyan). Scale bar =
50μm; D, dorsal; M, medial. B. Protocol for intracellular recordings from appendicular MNs.
Stage 52 MNs were identified by prior rhodamine dextran amine (RDA) retrograde labeling |
